A POINT MUTATION IN G-ALPHA(O) AND G-ALPHA(I1) BLOCKS INTERACTION WITH REGULATOR OF G-PROTEIN SIGNALING PROTEINS

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydroly sis by G alpha subunits and are thought to be responsible for rapid de activation of enzymes and ion channels controlled by G proteins, We wa nted to identify and characterize G(i)-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in t he yeast G alpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P, R,, Garrison, T, R,, Apanovitch, D, M., Hoffman, G,, Shue y, D, J,, Mason, R,, Cockett, M. I., and Dohlman, II, G, (1998) J. Bio l Chem. 273, 5780-5784), site-directed mutagenesis of alpha(o) and alp ha(i1) was done. G184S alpha(o) and G183S alpha(i1) show kinetics of G DP release and GTP hydrolysis similar to wild type. In contrast, GTP h ydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7, Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activ ated wild type alpha(o) and alpha(i1) to compete with fluorescein isot hiocyanate-alpha(o) binding to glutathione S-transferase-RGS4, The G - -> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in a lpha(o) and alpha(i1) that prevents RGS binding and GTPase activating activity, These mutant subunits should be useful in biochemical or exp ression studies to evaluate the role of endogenous RGS proteins in G(i ) function.